Assays capable of detecting and quantifying the presence of a particular nucleic acid molecule in a sample are of substantial importance in forensics, medicine, epidemiology and public health. Such assays can be used, for example, to identify the causal agent of an infectious disease, to predict the likelihood that an individual will suffer from a genetic disease, to determine the purity of drinking water or milk, or to identify tissue samples. The desire to increase the utility and applicability of such assays is often frustrated by assay sensitivity. Hence, it would be highly desirable to develop more sensitive detection assays.
Nucleic acid detection assays may be predicated on any characteristic of the nucleic acid molecule, such as its size, sequence and, if DNA, susceptibility to digestion by restriction endonucleases. The sensitivity of such assays may be increased by altering the manner in which detection is reported or signaled to the observer. Thus, for example, assay sensitivity may be increased through the use of detectably labeled reagents. A wide variety of such labels have been used for this purpose. Detectable labels include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels.
Although the use of highly detectable labeled reagents can improve the sensitivity of nucleic acid detection assays, the sensitivity of such assays remains limited by factors including, but not limited to, non-specific reactions which increase the background signal and limitations in discriminating between sequences which differ by one or a few nucleotides. In response to these problems, a variety of detection and quantification methods using DNA amplification have been developed.
Amplification of DNA by the polymerase chain reaction (PCR) uses a pair of primers whose sequences determine the specificity of the amplification. However, the amplicon sequence outside of the priming regions has little or no contribution to amplification specificity. In a typical PCR reaction, forward and reverse primers are used to preferentially target and amplify a DNA sequence of interest. In TaqMan® assays, a probe picked from within the amplicon sequence is also provided to selectively detect and monitor the amplification product. The remainder of the target sequences outside of the primer and probe regions is not utilized. A need still remains to increase the specificity of amplification reactions, especially for amplification of samples which differ by one or a few nucleotides. Examples of such analyses include, but are not limited to, genotyping, rare allele detection, and pre-amplification of mutant sequences.